5048
J. Org. Chem. 1993,58, 5048-5049
Triplex Formation by an Oligonucleotide Containing Na-(3-Acetamidopropyl)cytosine Chin-Yi Huang, Cynthia D. Cushman, and Paul S. Miller’ Department of Biochemistry, School of Hygiene and Public Health, The Johna Hopkina University, 615 North Wolfe Street, Baltimore, Maryland 21205 Received May 28, 19938
Summary: Triplex formation has been observed between an oligodeoxyribonucleotideduplex and an oligodeoxyribopyrimidine which contains N4-(3-acetamidopropyl)cy-
d-CTTCTTTTTTXTTTT I d - GAAGAAAAAAYAAAA I1 CTTCTTTTTTZTTTT-d I11
tosine, a novel base analog which selectively interacts with a C-G base pair of the target duplex.
Oligodeoxyribopyrimidinescan interact with the homopurine strand of a n oligodeoxyribopurine-oligodeoxyribopyrimidine duplex through the formation of C+*G.Cand T-A-Tbase triads.14 The presence of a single COGor T-A base pair in the otherwise purinepyrimidine sequence motif of the target duplex can be accommodated by naturally occurring bases in the third strand, although such “mismatches” generally lead to overall reduced stability of the triplex.= An exception to this is the observation that G can recognize a single T*A base pair.1fa9316-73 Recently, Griffii et al. described a nonnatural base, 4-(3-benzamidophenyl)imidazole,which interacts selectively with COGand T-A base pair~.~JO 2’-Deoxynebularine, when incorporated into a third-strand oligopyrimidine, has been reported to bind to C-G base pairs and to also interact with A.T base pairs of a target dup1ex.l’ We have studied triplex formation in the oligodeoxyribonucleotide system shown in Figure 1. Oligomer I(X) contains thymine, 5-methylcytosine, C, and N4-modified cytosine, X. The structures of the modified bases are shown in the lower part of Figure 1. Oligomerscontaining N4-(butyl)C,I@),N4-(3-~arboxypropyl)C,I(3), or N4-(3aminopropyl)C,I(4),were prepared by reaction of oligomer I( 1) with the appropriate amine in the presence of sodium bisulfite. This reaction selectively transaminates cytosine but does not modify 5-methylcytosines because 5-methylcytosine, unlike cytosine, does not form an adduct with bisulfite.12 Oligomer I(5), which contains an N4-(3acetamidopropyl)C,was prepared by reaction of 1(4) with
1 2 3 4 5
88,9397-9401.
( 6 ) Yoon, K.;Hobbs, C. A.; Koch, J.; Sardaro, M.; Kutny, R.; Weis, A. L. hoc. Natl. Acad. Scr. U.S.A.1992,30, 389-3844. (6) Miller, P. 5.;Cushman, C. D. Biochemurtry 1993,32,2999-3004. (7) Griffin, L.; Dervan, P. B. Science 246, 967-971. (8) Wang, E.; Malek,S.; Feigon, J. Biochemistry 1992,31,4838-4846. (9) Kiessling, L. L.; Griffin, L. C.; Dervan, P. B. Biochemistry 1992,
31, 2829-2834. (10) Griffin,L. C.; Kiessling, L. L.; Beal,P. A.; Gillespie, P.; Dervan, P. B. J. Am. Chem. Soc. 1992,114,7976-7982. (11) Stilz, H.U.; Dervan, P. B. Biochembtry 1993,32,2177-2186. (12) Miller, P. 5.;Cushman,C. D. Bioconjugate Chem. 1992,3,74-79.
5
Figure 1. Sequences of the triplex-formingoligodeoxyribonucleotides I, 11,and 111.Cis 5-methylcytosine,and the structures of the “-modified cytosines, X, are shown below the sequences.
0.34
0.32 0
1
U
0.30 0.28
0
Abstract published in Aduance ACS Abstracts, September 1,1993. (1)See, for example: (a) LeDoan, T.; Perrouault, L.; Prawuth, D.; Habhoub, N.;Decout,J.-L.; Thuong, N. T.; Lhomme,J.; Helene,C.Nucleic Acids Res. 1987,15,7749-7760. (b) Moser, H. E.; Dervan, P. B. Science 1987,238, 616-660. (c) Francois, J.-C.; Saison-Behoaras, T.; Barbier, C.; Chaseigonol, M.; Thuong, N. T.; Helene, C. Roc. Natl. Acad. Sci. U.S.A. 1988,16,11431-11440. (d) Rajagopal, P.; Feigon, J. Nature 1989, 339,637-640. (e) Rajagopal, P.; Feigon, J. Biochemistry 1989,28,78597870. (f) de 10s Santos, C.; Roeen, M.; Patel, D. Biochemistry 1989,28, 7282-7288. (9) Radh+rishnan, I.; Gao, X.;de loa Santos, C.; Live, D.; Patel, D. J. Wochemrstry 1991,30,9022-9030. (2) Macaya, R. F.; Gilbert, D. E.; Malek, S.; Sinsheimer, J. S.; Feigon, J. Science 1991, 254, 270-274. (3) Mergny, J.-L.; Sun, J.-S.; Rougee, M.; Montenay-Garestier, T.; Barcelo, F.; Chomilier,J.; Helene, C. Biochemistry 1991,30,9791-9798. (4) Roberta,R. W.; Crothera,D. M.Proc. Natl. Acad. Sci. U.S.A. 1991, 0
R - H R = -(CH2),CH3 R = - (CH,),COOH R -(CH2)3NH2 R - (CH2),NHC(0)CH3
10
20
30
40
50
60
Temp ‘C
Figuret Melting profile ofI.II.III(S.C.G).The buffer contained 0.1 M sodium chloride, 20 mM magnesium chloride, 50 mM Tris, pH 7.0, and the strand concentration waa 1 rM per strand. The solution waa heated at 0.5 OC/min.
p-nitrophenyl acetate in acetonitrile solution in the presence of triethylamine. Control experiments showed that thistreatment does not modify 5-methyldeoxycytidine nor does it acetylate the 5‘- or 3‘-hydroxyl groups of the nucleoside. The modified oligomers were completely hydrolyzed by treatment with a combination of snake venom phosphodiesterase and bacterial alkaline phosphatase and gave the expected nucleosides, including the appropriate C-modified nucleoside.12 Interactions between oligomer I(X) and duplex 11-111(YoZ)were studied by recording the Am us temperature profiie over the temperature range 0-60 OC. These melting experiments were carried out in a buffer containing 0.1 M sodium chloride, 20 mM magnesium chloride, and 50 mM
0022-3263/93/1958-504&3$04.00/0 0 1993 American Chemical Society
Communications
J. Org. Chem., Vol.58,No. 19, 1993 5049
Table I. Melting Temperatures of the Third Strands of Triplex 1.11411 base substitutions'
X
Y
2
1
C C C C
G G G G G
T A G G
A T C C
2 3 4
5
5 5 5 1
C
Tmb("C) strand I
